Kelly Hypoxia RNA-Seq
RNA-Seq of Hif1a, Hif2a & Hif1b gene knock-outs
1.) Data processing
combine sample list
| experiment | RNAs | conditions | date | seq_id | Seq_runs | |
|---|---|---|---|---|---|---|
| 3 | Katharina | 16 | Kelly_Nx Kelly_Hx HIF1A_Hx HIF2A_Hx | 2018-09-13 2018-09-14 | P557 | 16 |
| 1 | Simon | 22 | Kelly_Nx Kelly_Hx HIF1A_Nx HIF1A_Hx HIF1B_Nx HIF1B_Hx | 2017-05-04 2021-06-16 2021-08-25 2021-08-27 | P2041 | 22 |
| 2 | Ulrike | 50 | Kelly_Nx Kelly_Hx HIF1A_Nx HIF1A_Hx HIF2A_Nx HIF2A_Hx | 2023-06-02 2023-06-08 2023-06-15 2023-06-28 | P3302 | 150 |
DESeq2 Design
design = ~experiment+genotype+treatment+genotype:treatment
MA plot & Dispersion
Transformations
Sample distance
Principal component analysis
Plot example counts
Contrasts_overview
## cutoffs ## differential expressed: p=0.05,bM=10,l2FC=1 ## top genes: p=0.01,bM=100,l2FC=2
| all.DEGs | top.DEGs | |
|---|---|---|
| 1 | ||
| deg_Hif1a.Hx.vs.Nx | 6166 | 983 |
| deg_Hif2a.Hx.vs.Nx | 3394 | 542 |
| deg_Hif1b.Hx.vs.Nx | 2051 | 310 |
| deg_Kelly.Hx.vs.Nx | 5301 | 866 |
| 2A Nx | ||
| deg_Nx.Hif1a.vs.Kelly | 223 | 21 |
| deg_Nx.Hif2a.vs.Kelly | 596 | 75 |
| deg_Nx.Hif1b.vs.Kelly | 721 | 58 |
| 2B Hx | ||
| deg_Hx.Hif1a.vs.Kelly | 1013 | 77 |
| deg_Hx.Hif2a.vs.Kelly | 2532 | 348 |
| deg_Hx.Hif1b.vs.Kelly | 4208 | 531 |
| deg_Hx.Hif2a.vs.Hif1a | 4174 | 607 |
| deg_Hx.Hif1b.vs.Hif1a | 4972 | 643 |
| deg_Hx.Hif1b.vs.Hif2a | 2043 | 249 |
| 3 interaction | ||
| deg_Hif1aHxNx.vs.KellyHxNx | 686 | 54 |
| deg_Hif2aHxNx.vs.KellyHxNx | 2238 | 281 |
| deg_Hif1bHxNx.vs.KellyHxNx | 3887 | 431 |
| group contrasts | ||
| deg_Hx.Hif1b.vs.Hif12a | 2741 | 224 |
| deg_Hx.Kelly.vs.allHIFs | 1502 | 148 |
| deg_Hx.vs.Nx | 3599 | 495 |
Which results to use?
Example for Hif1a
Results 1
SCARB1 doesn’t fit!
Kelly.Hx.vs.Nx
| baseMean | log2FoldChange | lfcSE | stat | pvalue | padj | symbol | |
|---|---|---|---|---|---|---|---|
| ENSG00000073060 | 11958.596 | 1.912261 | 0.0564790 | 33.85793 | 0 | 0 | SCARB1 |
| ENSG00000132382 | 6908.702 | -1.840556 | 0.0836373 | -22.00641 | 0 | 0 | MYBBP1A |
| ENSG00000186469 | 8085.827 | 1.871876 | 0.0904267 | 20.70048 | 0 | 0 | GNG2 |
Hif1a.Hx.vs.Nx
| baseMean | log2FoldChange | lfcSE | stat | pvalue | padj | symbol | |
|---|---|---|---|---|---|---|---|
| ENSG00000073060 | 11958.596 | 2.513715 | 0.0674164 | 37.28643 | 0 | 0 | SCARB1 |
| ENSG00000132382 | 6908.702 | -2.577358 | 0.1000207 | -25.76825 | 0 | 0 | MYBBP1A |
| ENSG00000186469 | 8085.827 | 2.579589 | 0.1081358 | 23.85510 | 0 | 0 | GNG2 |
compare results with contrast vsvs (Hif1a Hx vs. Nx VS. Kelly Hx vs. Nx
